Interest in population-screening programs for abnormal hemoglobins in the United States has increased primarily because of the National Sickle Cell Disease Program developed by the Department of Health, Education and Welfare. Thus, screening for hemoglobinopathies has become very important within the past five years. For example, many hospitals now screen all incoming surgical patients for such abnormalities. Because electrophoresis on cellulose acetate is the primary procedure used in the Federal Sickle Cell Disease Program, cellulose acetate electrophoresis at pH 8.4-9.2 currently is the most common electrophoretic screening method for hemoglobinopathies. It has been found, however, that cellulose acetate cannot adequately separate certain combinations of hemoglobin variants. See, for example, E. J. Hicks and D. J. Hughes, Clin. Chem., 21, 1072 (1975). Thus, in questionable cases, one must proceed to a secondary procedure such as electrophoresis (electrochromatography) on citrate agar to obtain a more definitive diagnosis.
The use of citrate agar in the electrophoresis of hemoglobin variants has been summarized by R. J. Wieme, "Agar Gel Electrophoresis", Elsevier Publishing Company, Amsterdam, 1965. The typical citrate agar procedure involves the use of a gel buffer having a citrate concentration of 0.05 and a pH of 6.2, with only minor variations, if any, in pH or citrate concentration. See, for example, C. N. LeCrone et al., Clin. Chem., 22, 1743 (1976).
The hemoglobin variants separated by the citrate agar electrochromatography procedure can be estimated visually, scanned by a densitometer to determine the relative amounts of each variant present, or stained to enhance visualization or densitometry readings of the hemoglobin variants. Typical staining procedures, however, involve hemoglobin-specific dyes such as benzidine or o-dianisidine. Benzidine is a known carcinogen and has been removed from the market by the Federal Food and Drug Administration. The o-dianisidine is a benzidine derivative and is suspected to be carcinogenic as well; thus, such compound also may be removed from the market. In addition, these stains are light sensitive and darken upon storage. Furthermore, the prior art agar gels must be stored wet and thus can be bulky and difficult to store.